Chimeric antisense oligonucleotides and cell transfecting formulations thereof

ABSTRACT

Chimeric oligonucleotide of the formula 5′-W-X 1 -Y-X 2 -Z-3′, where W represents a 5′-O-alkyl nucleotide, each of X 1  and X 2  represents a block of seven to twelve phosphodiester-linked 2′-O-alkyl ribonucleotides, Y represents a block of five to twelve phosphorothioate-linked deoxyribonucleotides, and Z represents a blocking group effective to block nuclease activity at the 3′ end of the oligonucleotide, are described. These compounds exhibit high resistance to endo- and exonucleases, high sequence specificity, and the ability to activate RNAse H, as evidenced by efficient and long-lasting suppression of target mRNA. The oligonucleotides are preferably transfected into cells in formulations containing a lipid-peptoid conjugate carrier molecule of the formula  
     L-linker-[N(CH 2 CH 2 NH 2 )CH 2 (C═O)-N(CH 2 CH 2 R)CH 2 (C═O)—N(CH 2 CH 2 R)CH 2 (C═O)] 3 -NH 2 ,  
     where L is a lipid moiety, including a steroid, and each group R is independently selected from alkyl, aminoalkyl, and aralkyl.

[0001] This application claims priority to U.S. provisional applicationSer. No. 60/151,246, filed Aug. 27, 1999, which is hereby incorporatedby reference in its entirety and for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to antisense oligonucleotides, andmore particularly to chimeric antisense oligonucleotides which exhibithigh resistance to endo- and exonucleases, high sequence specificity,and the ability to activate RNAse H, as evidenced by efficient andlong-lasting knockout of target MRNA. Also provided are formulations ofthe oligonucleotides with carrier molecules which provide efficienttransfection into cells.

BACKGROUND OF THE INVENTION

[0003] The use of antisense oligonucleotides to specifically inhibit thefunction of targeted genes has been the subject of extensive research,due to its promise in selective antiviral and anticancer therapy. Manystudies have been directed to the design of oligonucleotide analogshaving an optimal combination of properties, including stability (i.e.resistance to cellular nucleases), cellular uptake, DNA/RNA bindingaffinity and specificity, and efficiency of inhibition. Because thephosphodiester linkages of native nucleic acids are degraded by endo-and exonucleases, many early studies were directed to designingnuclease-resistant analogs. Phosphorothioates are one such class ofcompounds, which are relatively stable in vivo and retain the ability toactivate RNAse H, the primary mechanism by which antisenseoligonucleotides deactivate target RNA. However, the use ofphosphorothioates presents several disadvantages, including a high levelof non-specific binding to other cellular components, often leading tounwanted side effects, and reduced binding affinity for RNA.

[0004] Oligomeric ribonucleotides substituted at the 2′-oxygen show highRNA binding affinities and, in comparison to the unsubstitutedribonucleotides, reduced sensitivity to endogenous nucleases. Although2′-O-methyl substituted ribonucleotides provide greater binding affinitythan those having larger substituents (e.g. ethyl, propyl, pentyl,allyl), the larger substituents are reported to confer greaterexonuclease resistance (see, for example, Monia et al., J. Biol. Chem.271(24): 14533, 1996). Arrow et al. (U.S. Pat. No. 5,849,902) statedthat “2′-O-methyl bases with phosphodiester linkages are degraded byexonucleases and so are not suitable for use in cell or therapeuticapplications of antisense.” Phosphorothioate and phosphotriesterlinkages were recommended by the latter group as having greaterstability, even though they presented the disadvantages of reducedbinding affinity, more difficult synthesis (phosphotriester) and/orgreater toxicity (phosphorothioate).

[0005] Therefore, there is still a need for antisense oligonucleotidecompositions with optimal combinations of antisense activity, targetbinding affinity, biocompatibility, and stability.

SUMMARY OF THE INVENTION

[0006] The present invention includes, in one aspect, a chimericoligonucleotide having the formula 5′-W-X¹-Y-X²-Z-3′, where W representsa 5′-O-alkyl nucleotide, such as a 5′-O-alkyl thymidine; each of X¹ andX² represents a block of seven to twelve phosphodiester-linked2′-O-alkyl ribonucleotides; Y represents a block of five to twelvephosphorothioate-linked deoxyribonucleotides; and Z represents ablocking group effective to block nuclease activity at the 3′ end of theoligonucleotide. In one embodiment, Z is a 3-to-3′ linked nucleotide. Infurther embodiments, the alkyl groups of the 5′-O-alkyl nucleotideand/or the 2′-O-alkyl ribonucleotides are methyl groups. In stillfurther embodiments, groups W and/or Z are linked to X¹ and X²,respectively, via phosphodiester linkages, phosphotriester,phosphorothioate, or phosphoramidate linkages. Preferably, W is linkedvia a phosphodiester or phosphorothioate linkage, and Z is linked via arelatively nuclease-resistant linkage; i.e. a phosphotriester,phosphorothioate, or phosphoramidate linkage.

[0007] In specific embodiments, the segment X¹-Y-X² of the chimericoligonucleotide has a sequence represented by any of SEQ ID NOs: 1-24disclosed herein.

[0008] In another aspect, the invention provides a therapeuticcomposition which comprises an oligonucleotide as described above in apharmaceutically acceptable vehicle. In preferred embodiments, thevehicle includes a lipid-cationic peptoid conjugate or “lipitoid”. Oneclass of lipid-cationic peptoid conjugates includes compounds of theformula:

L-linker-[N(CH₂CH₂NH)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)]₃-NH₂,

[0009] where the lipid group L is a fatty acid-derived group, such as aphospholipid group (i.e. ROOCCH₂CH(COOR)CH₂OP(O)₂O-), having fatty alkylor alkenyl chains between about 8 and 24 carbon atoms in length, or asteroid-derived group, such as a cholesteryl group, and the portion ofthe molecule to the right of the linker is the peptoid segment. In thepeptoid segment, R is selected from alkyl (branched or unbranched),aminoalkyl, and aralkyl. As used herein, “aralkyl” refers to an alkyl,preferably lower alkyl, substituent which is further substituted with anaryl group; one example is a benzyl group. “Aryl” refers to asubstituted or unsubstituted monovalent aromatic radical having a singlering (e.g., benzene) or two condensed rings (e.g., naphthyl). This termincludes heteroaryl groups, which are aromatic ring groups having one ormore nitrogen, oxygen, or sulfur atoms in the ring, such as furyl,pyrrole, pyridyl, and indole. By “substituted” is meant that one or morering hydrogens in the aryl group is replaced with a substituent,preferably selected from a halide, a lower alkyl or lower alkoxy group,halomethyl, or haloethyl.

[0010] In specific embodiments, R is isopropyl or 4-methoxyphenyl. Asingle lipitoid may include different groups R, or they may be the samewithin the molecule.

[0011] The linker may be a direct bond, or it may be a substantiallylinear linking group, such as an oligopeptide or an alkyl chain, of anyeffective length. The linker may also be an alkyl chain having one ormore heteroatom-containing linkages, selected from the group consistingof ester, amide, carbonate, carbamate, disulfide, peptide, and ether, ateither terminus of the chain or intervening between alkyl bonds. Inselected embodiments, the linker is from 2 to about 30 atoms, or from 3to about 15 atoms, in length.

[0012] In another aspect, the invention provides a method of inhibitingexpression of a target gene in a subject, which comprises administeringto the subject, in a pharmaceutically acceptable vehicle, an amount of achimeric oligonucleotide effective to specifically hybridize to all orpart of a selected target nucleic acid sequence derived from the gene,where the chimeric oligonucleotide has a structure as described above.In one embodiment, the target nucleic acid sequence is a MRNA derivedfrom the target gene. In specific embodiments, the segment X′-Y-X² ofthe chimeric oligonucleotide has a sequence represented by any of SEQ IDNOs: 1-24 disclosed herein. In further embodiments, the vehicle includesa lipid-cationic peptoid conjugate such as described above.

[0013] As shown herein, the chimeric oligonucleotides of the inventionprovided surprisingly high stability and efficient and long-lastingknockout of target mRNA. These and other objects and features of theinvention will become more fully apparent when the following detaileddescription of the invention is read in conjunction with theaccompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 shows a schematic representation of a chimericoligonucleotide, in accordance with one embodiment of the invention;

[0015]FIG. 2 shows a selection of phospholipid-peptoid conjugates(“lipitoids”) and cholesterol-peptoid conjugates (“cholesteroids”)useful as oligonucleotide carriers in compositions and methods of theinvention;

[0016]FIG. 3 shows the effect on AKT1 mRNA level of antisense oligos toAKT1 delivered to HT1080 cells via Effectene™, Lipitoid 1, and peptoid1, and control oligos (AKT2-AS, AKT2-RC, and AKT1-RC) delivered viaEffectene™;

[0017]FIG. 4 shows the effect on AKT1 mRNA level of antisense oligos toAKT1 delivered to colon cancer cells (Lovo) in conjunction with:Lipitoid 1, two different charge ratios of Lipitoid 2(DMPE(NaeNiaNia)₃), two different charge ratios of Cholesteroid 1(Chol-β-ala-(NaeNmpeNmpe)₃), and the commercially available transfectionagent Cytofectin™;

[0018] FIGS. 5-7 show the effects on cell proliferation of transfectionof Lovo, Kml2L4, and Colo320DM colon cancer cells, respectively, withchimeric oligonucleotides of the invention, in conjunction withdifferent lipitoid and cholesteroid carriers; and

[0019]FIG. 8 shows the results of several cell viability assays onKml2L4 and HCT-166 cells transfected with oligonucleotides inconjugation with lipitoids 1 and 2, cholesteroids 1 and 3, and thecommercially available transfection agents Lipofectin® and Cytofectin™,where the white regions indicate levels of healthy cells.

DETAILED DESCRIPTION OF THE INVENTION

[0020] I. Chimeric Antisense Oligonucleotides

[0021] A. Structure

[0022] The chimeric oligonucleotides of the invention have the generalstructure shown below:

5′-W-X¹-Y-X²-Z-3′

[0023] In this structure, the central portion of the molecule,represented by Y, is a block of between five and twelvephosphorothioate-linked deoxyribonucleotides (phosphorothioate DNA, orPS DNA). In one embodiment, the block Y is effective to activate RNAse Hwhen hybridized to a sufficiently complementary strand of RNA, thuspromoting cleavage of the RNA. Block Y is flanked by two binding blocks,represented by X¹ and X², each having between seven and twelvephosphodiester-linked 2′-O-alkyl ribonucleotide subunits (phosphodiester2′-O alkyl RNA, or PO 2′-O-alkyl RNA). As used herein, “alkyl” refers toa fully saturated acyclic monovalent radical containing carbon andhydrogen, which may be branched or a straight chain; examples of alkylgroups are methyl, ethyl, n-butyl, t-butyl, n-heptyl, and isopropyl.“Lower alkyl” refers to an alkyl radical of one to six carbon atoms, andpreferably one to four carbon atoms.

[0024] The alkyl groups of the 2′-O-alkyl ribonucleotide subunits arepreferably lower alkyl groups. In one embodiment, the alkyl groups aremethyl groups, which provide generally superior binding and cellularuptake in comparison to longer alkyl groups. The binding blocks, whilenot necessarily effective to participate in activation of RNAse H,provide binding affinity to sufficiently complementary RNA strands andmay also provide reduced cellular toxicity compared tophosphorothioate-linked subunits.

[0025] Blocking groups Z and W are provided at the 3′ and 5′ termini,respectively. In one embodiment, the groups W and Z are linked to therespective X blocks by phosphodiester linkages; in another embodiment,they are attached via phosphorothioate linkages. The 3′-blocking group Zis preferably a 3′-to-3′ linked nucleotide, although this terminus mayalso be blocked by other methods, e.g. by attachment of the terminalnucleotide via a relatively nuclease-stable linkage (e.g.phosphorothioate, phosphoramidate, phosphotriester) or appendage of anon-nucleotide moiety.

[0026] The 5′-terminus is blocked with a 5′-O-alkyl nucleotide subunit(W), where alkyl is preferably lower alkyl. In one embodiment, W is a5′-O-methyl thymidine. This blocking group is found to confer stabilityto the chimeric oligonucleotides in cell culture and in serum. Forexample, the duration of mRNA knockout in cell cultures (discussedfurther below) typically ranged from 3-5 days post transfection. Inaddition to providing stability, this blocking group, and the 3′-to-3′nucleotide blocking group, were found not to interfere with uptake ordistribution of the oligonucleotides.

[0027] The chimeric oligonucleotides of the invention can be preparedusing solution phase or, preferably, solid phase synthesis, according toestablished procedures. Synthesis of an exemplary chimericoligonucleotide, such as shown in FIG. 1, is described in Example 1.

[0028] B. Antisense Activity

[0029] Antisense chimeric oligonucleotides based on the formula above,having sequences directed against AKT1 (SEQ ID NO: 1) or AKT2 (SEQ IDNO: 2), were prepared as described in Example 1. The oligonucleotidesalso included a 5′-terminal 5′-O-methyl thymidine, as indicated by theformula above. In these oligonucleotides, X¹ and X² were seven-baseblocks of 2′-O-methyl PO RNA, Y is PS DNA, Z was a 3′-to-3′ linkednucleotide, and W was a 5′-O-methyl thymidine. Both Z and W were linkedto the respective X blocks via phosphodiester linkages.

[0030] When transfected into cells as described in Example 2, chimericantisense oligonucleotides of the invention having various sequences(see Table 1) showed surprisingly effective degradation of endogenousmRNA, resulting in a loss of activity of the respective genes. FIGS. 5and 6 show levels of endogenous AKT1 mRNA in colon cancer cells andHT1080 cells, respectively, transfected with anti-AKT1 chimericoligonucleotides (SEQ ID NO: 1). Similarly, a chimeric antisenseoligonucleotide directed against hCHK1 (SEQ ID NO: 3) showed degradationof endogenous mRNA, loss of chk1 kinase activity, and loss of chk1function (i.e. G2 cell cycle checkpoint control).

[0031] Additional chimeric oligonucleotides having the sequences shownin Table 1 were prepared and administered to cells as described inExample 2. (Each oligonucleotide included a 5′-O-methyl thymidine, asdescribed above, which is not shown in the listed sequences.) In theseoligonucleotides, with reference to the formula above, X¹ and X² areseven-base blocks of 2′-O-methyl PO RNA, Y is a mine- to eleven-baseblock of PS DNA, Z is a 3′-to-3′ linked nucleotide, and W is a5═-O-methyl thymidine. Both Z and W are linked via phosphodiesterlinkages.

[0032] With the exception of the mTyr oligo (SEQ ID NO: 17-18), whichwas transfected into B16 melanoma cells, all oligos shown in Table 1were transfected into HT1080 cells, using “Lipitoid 1” (see below) fortransfection, and incubated for 24 hours. The table gives theapproximate level of mRNA knockout observed in each case. Reduction inmRNA levels of 90% or more were frequently observed, as shown in theTable. TABLE 1 SEQ Nucleotide ID Antisense Oligonucleotide Position inLocus/Acc for mRNA NO: Sequence (X¹—Y—X² Segment) Gene Gene Genbankknockout 1 CCATAGTGAGGTTGCATCTGGTGCC AKT1 AKT1-2074 NM005163 >90% 2GTTCCCTTGCCAAGGAGTTTGAGAT AKT2 AKT2-548 NM001626 >85% 3CCCAGAGCCGATGGTCCGATCATCT CHK1 CHK1-1460 CHEK1 >90% 4GACCCACTTCCCTGAAAATCCGAAA CHK2 CHK2-430 AF086904 >90% 5CGGCGTTTTCCTTTCCCTACAAGC CHK2-518 AF086904 6 AGCGGCAGAAGTTGAGGTATGTTGACK1E CK1E-766 HUMCSNK1E >80% 7 CCTGCCAGTATGAAGTTGGGAAGCG E1AF E1AF-1729HUME1AF >90% 8 GCGAAGTCCGTCTGTTCCTGTTTGA E1AF-710 HUME1AF 9TCTTCCTCACAGACCTTCGGGCAAG IGFR1 IGF1R-1025 HSIGFIRR >80% 10TGCTGATAGTCGTTGCGGATGTCG IGF1R-156 HSIGFIRR 11 GTGTTTGTTCAGGGTTCCATTTCGGILK ILK-687 HSU40282 >90% 12 GCATGTGGAAGGTAGGGAGGCAAGA KRAS KRAS-2576HUMKRASM >85% 13 ACCATATACCCAGTGCCTTGTGCGG KRAS-3352 HUMKRASM 14GAAGCCCCACTTGCGGTCGTCAT MMP2 MMP2-1098 HUMCN4GEL >80% 15ACGAGCAAAGGCATCATCCACTGTC MMP2-367 HUMCN4GEL 16GCTTTCTCTCGGTACTGGAAGACGT MMP9 MMP9-2007 HUM4COLA >80% 17AACCCATGAAGTTGCCTGAGCACTG mTyr MTYR-332 MUSTYR >90% 18TTTCAGGGTGACGACCTCCCAAGTA MTYR-814 MUSTYR 19 ATCTGGTCGCCTCATTTGCTCAACTp110α P110A-2205 HSU79143 >95% 20 TTTCTTCACGGTTGCCTACTGGTTC P110A-307HSU79143 21 TGATGAAGAGATTCCCATGCCGTCG p110β P110B-2980 S67334 >90% 22TGTAGTCTTTCCGAACTGTGTGGGC P110B-3181 S67334 23 CTGTGAGCAACAGCTGTCGTCGTCTPDK1 PDK1-1494 NM002613 >90% 24 GGCAGTCATTAGCAGGGTGATGGTG UPAR UPAR-1242HSU08839 >80%

[0033] II. Transfection Agents

[0034] A variety of strategies exist for delivery of nucleic acidcompositions to cells. Viral vectors provide relatively efficientdelivery, but in some cases present safety problems due to the risk ofimmunological complications or unwanted propagation in the subject.Adenoviral vectors have shown certain advantages in that they do notintegrate into the genome of the cell and can be transduced into restingcells. However, all of these vectors must be prepared by time consumingrecombinant DNA techniques. Oligonucleotides may also be delivered tocells via chemical transfection agents, which have been the subject ofmuch recent work. These agents include polycationic molecules, such aspolylysine, and cationic lipids. The liposomal composition Lipofectin®(Felgner et al., PNAS 84:7413, 1987), containing the cationic lipidDOTMA (N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride)and the neutral phospholipid DOPE (dioleyl phosphatidyl ethanolamine),is widely used. Any of these methods, as well as other methods such ascalcium phosphate mediated transfection, can be used to deliver theoligonucleotides of the invention, according to reported procedures.

[0035] One method of delivery involves the use of transfection agentsknown as “lipitoids” and “cholesteroids”, described, for example, inco-owned PCT publications WO 98/06437 and WO 99/08711 (Zuckermann etal.), based on U.S. Ser. Nos. 60/023,867, 60/054,743, and 09/132,808,which are hereby incorporated by reference. These lipid-cationic peptoidconjugates are shown in these references to be effective reagents forthe delivery of plasmid DNA to cells in vitro. It is shown herein thatsuch compounds efficiently deliver oligonucleotides into a variety ofprimary and tumor cell lines. The efficiency of delivery was assessed byfluorescence analysis of FITC-labeled oligonucleotides, or by monitoringmRNA levels after transfection of chimeric antisense oligonucleotides,as described further below.

[0036] Any of the carriers described in the above-referencedapplications are suitable for use in transfection of theoligonucleotides described herein. Further disclosure of steroids usefulfor incorporating into steroid-cationic peptoid conjugates is found inPCT publication WO 97/46223 (Fasbender et al.) and corresponding U.S.Pat. No. 5,935,936, which are hereby incorporated by reference.

[0037] These compounds may be prepared by conventional solution orsolid-phase synthesis. In one such procedure, as described in Zuckermannet al., cited above, the N-terminus of a resin-bound peptoid is acylatedwith a spacer such as Fmoc-aminohexanoic acid or Fmoc-β-alanine. Afterremoval of the Fmoc group, the primary amino group is reacted withcholesterol chloroformate to form a carbamate linkage, e.g. as shown inCholesteroids 2, 3, and 4 of FIG. 2. The product is then cleaved fromthe resin with trifluoroacetic acid and purified by reverse-phase HPLC.A fatty acid-derived lipid moiety, such as a phospholipid, may be usedin place of the steroid moiety, as also shown in FIG. 2. The steroid orother lipid moiety may also be linked to the peptoid moiety by otherlinkages, of any effective length, readily available to the skilledpractitioner. The linker is a chain up to about 30 bonds in length, andmore preferably up to about 15 bonds in length, though any effectivelength may be used. The chain is typically linear or substantiallylinear, although branched chains (including oligopeptides) and linkerscontaining intervening cyclic groups can also be used. The linkergenerally comprises alkyl (C—C) bonds and one or more functional groupssuch as ester, amide, carbonate, carbamate, disulfide, peptide or etherbonds. The linker may comprise multiple functional groups, as in asuccinate ester or polyether, or it may be an oligopeptide, preferably a2- to 10-mer, and more preferably a 2- to 5-mer. The steroid or lipidmoiety and peptoid segment can also be joined by a direct bond.

[0038] In certain embodiments, the linker incorporates one or more bondswhich are susceptible to cleavage under appropriate conditions in vivo;for example, hydrolyzable ester, carbonate, carbamate, or peptide bonds;disulfide bonds, which are cleavable in cellular compartments having asufficiently reducing environment; and peptide bonds, cleavable byendogenous peptidases. With respect to the latter, polypeptide linkershaving ten or fewer, or, in further embodiments, five or fewer peptidelinkages are contemplated, though longer linkers may also be used.

[0039] In particular embodiments, the lipid-cationic peptoid conjugatebelongs to a class of compounds having the formula:

L—(CH₂)_(n)—(C═O)—[N(CH₂CH₂NH₂)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)—N(CH₂CH₂R)CH₂(C═O)]₃—NH₂,

[0040] where L is selected from (i) a phosphatidylethanolamino group(i.e. ROOCCH₂CH(COOR)CH₂OP(O)₂O—CH₂CH₂NH₂—), having fatty alkyl oralkenyl chains between about 8 and 24 carbon atoms in length, and (ii) acholesteryl group linked to the adjacent —(CH₂)_(n)— segment by anester, amide or carbamate linkage; n is 1-5; and R is selected fromisopropyl and 4-methoxyphenyl. Representative structures of this class,shown in FIG. 2, are given the following designations herein: Lipitoid1, or L1 DMPE(NaeNmpeNmpe)₃ Lipitoid 2, or L2 DMPE(NaeNiaNia)₃Cholesteroid 1, or Chol 1 Chol-β-ala-(NaeNmpeNmpe)₃ Cholesteroid 2, orChol 2 Chol-Ahx-(NaeNmpeNmpe)₃ Cholesteroid 3, or Chol 3Chol-β-ala-(NaeNiaNia)₃ Cholesteroid 4, or Chol 4 Chol-Ahx-(NaeNiaNia)₃

[0041] As used herein, the term “lipitoid” may be used generically toinclude both lipitoids and cholesteroids, unless referring to aparticular Lipitoid, such as L1 or L2, above.

[0042] To prepare transfecting compositions, an aqueous solution of apeptoid, lipitoid or cholesteroid is formulated with theoligonucleotide, as described in Example 2A. The components arepreferably used in relative amounts such that there are at least two,and preferably two to four, positive lipitoid charges for every DNAnegative charge. The exact ratio of antisense oligonucleotide tolipitoid is preferably determined empirically for each cell type, but isgenerally in the range of 1.5-2 nmol lipitoid/μg antisenseoligonucleotide. Cells may be transfected as described above and inExample 2B.

[0043] The extent of delivery of FITC-labeled chimeric oligonucleotidesinto human fibrosarcoma (HT1080) cells was assessed via fluorescenceanalysis. (All oligonucleotides used in the subsequent studies werechimeric oligonucleotides as described for the studies represented inTable 1.) The sequence of the oligonucleotides was the reverse controlof PDK1 (SEQ ID NO: 25, the reverse of SEQ ID NO: 23) so that the effectof the oligonucleotides on the cells would be minimal. Theoligonucleotides were transfected via complexation with (a) acommercially available transfection agent, Effectene™, (b) the peptoid(NaeNmpeNmpe)₃ (peptoid 1), (c) Lipitoid 1, in a 1:4 charge ratio ofoligo to lipitoid, and (d) Lipitoid 1, in a 1:3 charge ratio. Incomparison to Effectene™, Lipitoid 1 gave a significantly highertransfection efficiency and higher degree of nuclear delivery of theoligonucleotide, as evidenced by fluorescence analysis of thetransfected cells. The higher lipitoid/oligo charge ratio (1:4; c) alsoappeared to be more effective than the 1:3 ratio.

[0044]FIG. 3 shows the reduction in endogenous AKT1 mRNA in HT1080 cellsresulting from transfection of a chimeric antisense oligonucleotide toAKT1, as described above (AKT1-AS), in comparison to controloligonucleotides AKT1-RC (RC=reverse control; SEQ ID NO: 26; reverse ofSEQ ID NO: 1), AKT2-AS, and AKT2-RC (SEQ ID NO: 27; reverse of SEQ IDNO: 2). The same oligos were also delivered by commercial lipids(Effectene™) and peptoids ((NaeNmpeNmpe)₃). The results, depicted inFIG. 3, show that L1-transfected AKT1 chimeric antisenseoligonucleotides gave the most pronounced reduction in the target mRNAlevel.

[0045] Efficiency of oligonucleotide delivery by cholesteroids has beenfound to be similar or superior to that of (non-steroid) lipitoids. Forexample, as shown in FIG. 4, delivery of the chimeric anti-AKT1 oligodescribed above by Cholesteroid 1 achieved a better AKT1 MRNA knockoutthan delivery by Lipitoid 1 or Lipitoid 2 in a colon cancer cell line(Lovo).

[0046] The cholesteroids provide the additional benefit of substantiallyreduced toxicity to cells in vitro. FIG. 5 shows a 4 day proliferationassay, conducted as described in Example 3, of Lovo colon cancer cellsfollowing transfection of 50-300 nM of oligonucleotides. (Again, reversecontrol PDK1 chimeric oligonucleotides, expected to be non-active, wereused.). These charts demonstrate the significant increase inproliferation and viability of the Lovo cells following anoligonucleotide transfection with Cholesteroids 2 and 3 (FIGS. 5B,D) ascompared with transfection with Lipitoids 1 and 2 (FIGS. 5A,C). Thiseffect is not limited to this cell type, and was also observed inproliferation assays of Kml2L4 colon cancer cells (FIG. 6) and Colo320DMcolon cancer cells (FIG. 7).

[0047] To further investigate the reduced toxicity of the cholesteroids,a FACS analysis of cells was carried out, following transfection (seeExample 4), to determine the number of necrotic (PI+), early apoptotic(annexin+), late apoptotic (annexin+/PI+) and healthy cells(annexin−/PI−). The white columns in FIG. 8 reflect the number ofhealthy cells, while colored portions of the bars (demarcated by shortline segments for clarity) represent dead or dying cells. The analysiswas performed on Kml2L4 (FIGS. 8A-B) and HCT116 cells (FIGS. 8C-D). Thepercentage of dying cells was determined 4 hours (FIGS. 8A,C) or 24hours (FIGS. 8B,D) post-transfection. While different cell types showdifferent sensitivity to the transfection, cells transfected withcholesteroids consistently contained the most healthy cells and showedthe lowest degree of cell death. This lower toxicity was also seen incomparison of cholesteroids with the commercially available lipidsLipofectamine® and Cytofectin™.

EXAMPLES

[0048] The following examples illustrate but are not intended in any wayto limit the invention.

Example 1 Synthesis of Chimeric poRNA-psDNA-poRNA Oligonucleotides

[0049] The chimeric oligonucleotides were prepared using solid phasesynthesis, according to established procedures. A PerSpective Biosystems(Framingham, Mass.) 8909 Synthesizer and an ABI 394 Synthesizer(ABI/Perkin-Elmer, Foster City, Calif.) were used for the RNA additionsand the phosphorothioate linked DNA additions, respectively. It is alsopossible to perform the synthesis using only one instrument with eightamidite reagent bottles. Unless otherwise noted, all reagent preparationand synthesis was performed using the manufacturers' standard protocols.

[0050] The 5′-CPG support column, 5′-O-methyl-RNA phosphoramidites,5′-O-methyl-dT-CE phosphoramidite, and sulfurizing reagent,3H-1,2-benzodithiole-3-one-2,2-dioxide, were all obtained from GlenResearch (Sterling, Va.).

[0051] To carry out a representative synthesis, the last seven bases ofthe desired sequence were entered into the 8909 Synthesizer, suppliedwith 2′-O-methyl-RNA phosphoramidites, the appropriate 5′-CPG column wasattached, and a 1 μmole-scale RNA synthesis was performed with the finalDMT on.

[0052] The column was then removed from the 8909 and attached to the ABI394. The sulfurizing agent was installed in position 15 (to replace theoxidizer), and a synthesis for the phosphorothioate middle section ofthe oligo was carried out, using the 1 μmole Sulfur program with thefinal DMT on.

[0053] The column was then removed and replaced on the 8909. The lastseven 2′-O-methyl RNA bases were added, using the 1 μmole RNA program,DMT on. Finally, the chain terminator, 5′-O-methyl-dT-CE (cyanoethyl)phosphoramidite, was added, using a 1 μmole DNA protocol modified toextend the coupling time to 300 seconds. The oligonucleotide was cleavedfrom the support, deprotected and gel purified using standard methods.

Example 2 Antisense Inhibition of Target RNA

[0054] A. Preparation of Transfection Mixture

[0055] For each transfection mixture, a carrier molecule, preferably alipitoid or cholesteroid, was prepared to a working concentration of 0.5mM in water, sonicated to yield a uniform solution, and filtered througha 0.45 μm PVDF membrane. The antisense oligonucleotide was prepared to aworking concentration of 100 μM in sterile Millipore water.

[0056] The oligonucleotide was diluted in OptiMEM™ (Gibco/BRL), in amicrofuge tube, to 2 μM, or approximately 20 μg oligo/ml of OptiMEM™. Ina separate microfuge tube, lipitoid or cholesteroid, typically in theamount of about 1.5-2 nmol lipitoid/μg antisense oligonucleotide, wasdiluted into the same volume of OptiMEMTm used to dilute theoligonucleotide. The diluted antisense oligonucleotide was immediatelyadded to the diluted lipitoid and mixed by pipetting up and down.

[0057] B. Transfection

[0058] Cells were plated on tissue culture dishes one day in advance oftransfection, in growth media with serum, to yield a density attransfection of 60-90%. The oligonucleotide/lipitoid mixture was addedto the cells, immediately after mixing, to a final concentration of100-300 nM antisense oligonucleotide. Cells were incubated with thetransfection mixture at 37° C., 5% CO₂ for 4-24 hours. After incubation,the transfection mixture was removed and replaced with normal growthmedia with serum.

[0059] Total RNA was extracted using the RneaSy™ kit (QuiagenCorporation, Chatsworth, Calif.), according to manufacturer's protocols.

[0060] C. Reverse Transcription

[0061] The level of target mRNA was quantitated using the RocheLightCycler™ real-time PCR machine. Values for the target mRNA werenormalized versus an internal control (e.g. beta-actin). For each 20 μlreaction, extracted RNA (generally 0.2-1 μg total) was placed into asterile 0.5 or 1.5 ml microcentrifuge tube, and water was added to atotal volume of 12.5 μl. To each tube was added 7.5 μl of abuffer/enzyme mixture, prepared by mixing (in the order listed) 2.5 μlH₂O, 2.0 μl 10× reaction buffer, 10 μl oligo dT (20 pmol), 1.0 μl dNTPmix (10 mM each), 0.5 μl RNAsin® (20u) (Ambion, Inc., Hialeah, Fla.),and 0.5 μl MMLV reverse transcriptase (50u) (Ambion, Inc.). The contentswere mixed by pipetting up and down, and the reaction mixture wasincubated at 42° C. for 1 hour. The contents of each tube werecentrifuged prior to amplification.

[0062] D. LightCycler™ Amplification of RT Reactions

[0063] An amplification mixture was prepared by mixing in the followingorder: 1× PCR buffer II, 3 mM MgCl₂, 140 μM each dNTP, 0.175 pmol eacholigo, 1:50,000 dil of SYBR® Green, 0.25 mg/ml BSA, 1 unit Taqpolymerase, and H₂O to 20 μl. (PCR buffer II is available in 10×concentration from Perkin-Elmer (Norwalk, Conn.). In 1× concentration itcontains 10 mM Tris pH 8.3 and 50 mM KCl. SYBR® Green (Molecular Probes,Eugene, Oreg.) is a dye which fluoresces when bound to double strandedDNA. As double stranded PCR product is produced during amplification,the fluorescence from SYBR® Green increases.)

[0064] To each 20 μl aliquot of amplification mixture, 2 μl of templateRT was added, and amplification was carried out according to standardprotocols.

Example 3 Cell Proliferation Assay

[0065] Cells were seeded into 96 well plates at a density of 5000 cellsper well. For a 4 day proliferation assay, 5 independent 96 well plateswere prepared, one for each day. After overnight incubation, cells weretransfected using the procedure described above. On each day of theproliferation assay, all medium was removed from one plate and frozen at−70° C. On day four, all plates were developed with the Quantos™ assaykit (Stratagene, La Jolla, Calif.) which determines the amount of DNA,and thus the number of cells, in each well.

Example 4 Cytotoxicity Assay

[0066] Cells were seeded in 35 mm dishes at 35000 cells/well and allowedto attach overnight. Cells were then transfected witholigonucleotide/lipid formulations at 50-300 nM and incubated for 4 or24 hours. Cells were harvested 12 hours later, including the mediumcontaining floating cells. Live cells were then stained with propidiumiodine (PI) to detect necrotic and apoptotic cells and counterstainedwith FITC-coupled Annexin V (which detects early and late apoptoticcells) according to the R&D Systems (Minneapolis, Minn.) ApoptosisDetection Kit instructions. The cells were then analyzed by FACSanalysis to determine the relative number of PI+, annexin V+, PI+annexin V+ and PI−/annexin V− cells. The results are expressed aspercent (FIG. 8).

[0067] While the invention has been described with reference to specificmethods and embodiments, it will be appreciated that variousmodifications may be made without departing from the invention.

It is claimed:
 1. A chimeric oligonucleotide having the formula5′-W-X¹-Y-X²-Z-3′, where W represents a 5′-O-alkyl nucleotide; each ofX¹ and X² represents a block of seven to twelve phosphodiester-linked2′-O-alkyl ribonucleotides; Y represents a block of five to twelvephosphorothioate-linked deoxyribonucleotides; and Z represents ablocking group effective to block nuclease activity at the 3′ end of theoligonucleotide.
 2. The oligonucleotide of claim 1 , wherein the alkylgroups of the 5′-O-alkyl nucleotide and the 2′-O-alkyl ribonucleotidesare lower alkyl groups.
 3. The oligonucleotide of claim 2 , wherein thealkyl groups of the 2′-O-alkyl ribonucleotides are methyl groups.
 4. Theoligonucleotide of claim 1 , wherein the 5′-O-alkyl nucleotide is a5′-O-alkyl thymidine.
 5. The oligonucleotide of claim 1 , wherein the5′-O-alkyl nucleotide is linked to X¹ via a phosphodiester linkage or aphosphorothioate linkage.
 6. The oligonucleotide of claim 1 , whereingroup Z is linked to X² via a linkage selected from the group consistingof a phosphotriester linkage, a phosphorothioate linkage, and aphosphoramidate linkage.
 7. The oligonucleotide of claim 1 , wherein Zis a 3-to-3′ linked nucleotide.
 8. The oligonucleotide of claim 1 ,wherein the segment X¹-Y-X² has a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 1-24.
 9. A composition useful forinhibiting expression of a target gene in a subject, comprising achimeric oligonucleotide as recited in claim 1 in a pharmaceuticallyacceptable vehicle.
 10. The composition of claim 9 , wherein the vehicleincludes a lipid-cationic peptoid conjugate of the formula:L-linker-[N(CH₂CH₂NH₂)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)]₃-NH₂where L is selected from a lipid moiety comprising at least one fattyalkyl or alkenyl chain between about 8 and 24 carbon atoms in length anda steroid; each group R is independently selected from alkyl,aminoalkyl, and aralkyl, and the linker is selected from the groupconsisting of a direct bond, an oligopeptide, a substantially linearalkyl chain from 2 to about 30 bonds in length, and a substantiallylinear chain from 2 to about 30 bonds in length consisting of alkylbonds and one or more linkages selected from the group consisting ofester, amide, carbonate, carbamate, disulfide, peptide, and ether. 11.The composition of claim 10 , wherein the linker is from 3 to about 15bonds in length.
 12. The composition of claim 10 , wherein said fattyalkyl or alkenyl chain is between about 14 and 24 carbon atoms inlength.
 13. The composition of claim 10 , wherein L is a phospholipidgroup, having two fatty alkyl or alkenyl chains between about 8 and 24carbon atoms in length.
 14. The composition of claim 10 , wherein L is acholesteryl group.
 15. The composition of claim 10 , wherein R isisopropyl or 4-methoxyphenyl.
 16. The composition of claim 10 , whereinthe lipid-cationic peptoid conjugate is of the formula:L-(CH₂)_(n)-(C═O)-[N(CH₂CH₂NH₂)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)-N(CH₂CH₂R)CH₂(C═O)]₃-NH₂,where L is selected from (i) a phosphatidylethanolamino group, havingfatty alkyl or alkenyl chains between about 8 and 24 carbon atoms inlength, and (ii) a cholesteryl group linked to the adjacent —(CH₂)_(n)—segment by an ester, amide or carbamate linkage; n is 1-5; and R isselected from isopropyl and 4-methoxyphenyl.
 17. The composition ofclaim 16 , wherein the lipid-cationic peptoid conjugate is selected fromthe group consisting of compounds represented herein as: (a) Lipitoid 1,or DMPE(NaeNmpeNmpe)₃; (b) Lipitoid 2, DMPE(NaeNiaNia)₃; (c)Cholesteroid 1, or Chol-β-ala-(NaeNmpeNmpe)₃; (d) Cholesteroid 2, orChol-Ahx-(NaeNmpeNmpe)₃; (e) Cholesteroid 3, or Chol-β-ala-(NaeNiaNia)₃;and (f) Cholesteroid 4, or Chol-Ahx-(NaeNiaNia)₃.
 18. A method ofinhibiting expression of a target gene in a subject, comprisingadministering to the subject, in a pharmaceutically acceptable vehicle,an amount of a chimeric oligonucleotide as recited in claim 1 which iseffective to specifically hybridize to all or part of a selected targetnucleic acid sequence derived from the gene.
 19. The method of claim 18, wherein the target nucleic acid sequence is a mRNA derived from thetarget gene.
 20. The method of claim 19 , wherein the segment X¹-Y-X² ofthe chimeric oligonucleotide has a nucleotide sequence selected from thegroup consisting of SEQ ID NOs: 1-24.
 21. The method of claim 18 ,wherein the vehicle includes a lipid-cationic peptoid conjugate asrecited in claim 11 .